Clinical and Molecular Characteristics of Megakaryocytes in Myelodysplastic Syndrome

Objective  Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established. Methods  Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared. Results  The more frequently mutated genes in MDS patients were TUBB1 (11.54%), VWF (8.65%), NBEAL2 (5.77%), and the most common point mutation was TUBB1 p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34 + CD61 + MKs (10.00 vs. 4.00%, p  = 0.012), and short overall survival (33.15 vs. 40.50 months, p  = 0.013). Further, patients with a higher percent of CD34 + CD61 + MKs (≧20.00%) had lower platelet counts (36.00 × 10 9 /L vs. 88.50 × 10 9 /L, p  = 0.015) and more profound emperipolesis ( p  = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34 + CD61 + MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay. Conclusion  High proportion of CD34 + CD61 + MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.


Introduction
Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells, characterized by ineffective hematopoiesis, single or multilineage dysplasia, and risk of progression to acute leukemia.Both morphological and functional defects of platelets have been observed in MDS patients, due to the dysplasia of megakaryocytes (MKs). 1,2Megakaryocytic proliferation/differentiation is a complex process that involves up/down expression of signaling molecules in the bone marrow (BM) microenvironment.Acquired or inherited mutations affecting the of MK development have been identified.RUNX1 mutation is associated with thrombocytopenia, leading to the increased CD34 expression on MKs.4][5][6] In general, CD34 expression on the megakaryocytic lineage is limited to promegakaryoblast or megakaryoblast and declines progressively throughout cell maturation. 7Recent evidence suggests that a high level of CD34/CD61 dual positive MKs (CD34 þ CD61 þ MKs) is associated with lower platelet count, cytogenetic abnormalities, and shorter survival. 8,9linically, low platelet count is often related to bleeding complications in MDS patients.However, because classic "platelet generating" MKs are altered by gene mutation, it is conceivable that platelet function abnormalities may play a role as well.A few studies have reported that, mainly with platelet activation and aggregation, many MDS patients demonstrated impaired platelet phenotypes and reduced functions. 10,11Although platelet transfusions have greatly reduced the incidence of major hemorrhagic complications, refractoriness to infused platelets becomes a major clinical problem.In this study, we will investigate the pathological characteristics of BM MKs and their relationship with driver gene mutation, aiming to find the potential molecular targets in MDS.

Patients
Patients with MDS were identified at Ruijin Hospital, Shanghai Jiao Tong University from May 2019 to December 2023.All study participants were diagnosed and classified according to the World Health Organization (WHO) 2022 classification. 12The prognostic impact was evaluated with the International Prognostic Scoring Systems-Revised (IPSS-R). 13eripheral blood (PB) collection and BM biopsies were performed on cases after obtaining written consent.Patient-matched germline reference samples such as oral mucosal cells and hair with hair follicles were also harvested.Clinical data related to age, blood count, and BM biopsy at diagnosis were collected from patients' medical records.The overall survival (OS) was evaluated as disease outcomes, and events were defined as death.All survival end points were censored at the date of last follow-up when progression or death was not observed.The research protocol was approved by the Ethic Committees of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.

Morphological Evaluation and Immunocytochemistry
All patients had representative BM biopsies, PB and BM aspirate smears available for evaluation.BM smears were stained with Wright's-Giemsa (Baso Diagnostics Inc, Zhuhai, China) and observed by light microscopy (BX41, Olympus Corporation, Tokyo, Japan).For morphologic dysplasia, features of dysmegakaryopoiesis had to be present in at least 10% of the cells of the respective lineage.Multilineage dysplasia involved at least 10% of the cells in two or more lineages.Immunohistochemical staining for CD34 (1:160 dilution; Dako, Glostrup, Denmark) and CD61 (1:100 dilution; Dako, Copenhagen, Denmark) in MKs was performed on formalin-fixed paraffin-embedded BM biopsy sections after heat-induced antigen retrieval using the avidin-biotin peroxidase technique.One hundred MKs or all MKs in the biopsy (if MKs <100) were were considered as high-level or positive, and cases with CD34 þ CD61 þ MKs <20% were considered as negative.
Enzyme-Linked Immunosorbent AssayBM samples were harvested from MDS patients.BM fluids were obtained by centrifugation of 3,500 Â g for 15 minutes.Levels of S100A9 proteins in the BM fluids were measured using human S100A9 enzyme-linked immunosorbent assaykits (R&D Systems) according to the manufacturer's instructions.

Platelet Phenotyping and Function Analysis
Washed platelets in Tyrode's buffer at a concentration of 3 Â 10 8 /mL was performed as previously described. 14Platelet aggregation was analyzed at 37°C using a Platelet Aggregation Profiler (Chrono-Log, Havertown, Pennsylvania, United States).Three independent experiments were performed to ensure the accuracy of the experimental results obtained.Washed platelets were preincubated with peptides (250 μM) at 37°C for 30 minutes, stimulated for 3 minutes with or without thrombin (0.1 U/mL) at 37°C, and immediately fixed with 1% paraformaldehyde.The fixed platelets were labeled with a PE-CD62P and FITC-Annexin V antibody (BD, Franklin Lakes, New Jersey, United States) at concentrations recommended by the manufacturers.The same conjugated nonspecific isotype IgG was used as a negative control.CD62P and Annexin V surface expression were analyzed by flow cytometry.All the samples were analyzed or stored properly within 2 hours of sampling as recommended to avoid significant artifacts in platelet analysis and the release of cell microparticles due to storage.

DNA Extraction and Targeted Next-Generation Sequencing of Megakaryocytes
BM mononuclear cells were obtained by centrifugation on a Ficoll-Hypaque at a density gradient of 1,500 Â g for 25 minutes and then washed three times in phosphatebuffered saline.Genomic DNA was isolated from bone marrow mononuclear cells and was extracted by Qiagen blood extraction kit (Qiagen, Hilden, Germany) following the manufacturer's protocol.DNA quality was assessed by agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, United States).Targeted amplicon-based NGS of up to 20 MK genes were performed.DNA samples were subjected to targeted genome sequencing using Illumina HiSeq2000.As previously described, 15 we established filters for the pathogenic versus nonpathogenic call algorithm to determine clinically actionable pathogenic alterations and to exclude benign variants or polymorphisms.

RNA Isolations and RNA-Sequencing of Megakaryocytes
Ten milliliters of fresh BM, which had been collected in EDTA tubes, was processed within 6 hours after collection and stored at 4°C.Nucleated BM cells were separated over a discontinuous Percoll gradient.After Percoll gradient centrifugation, the cells were washed with phosphate buffered saline and resuspended in RPMI 1,640 medium with 0.1% bovine serum albumin.The cells were stained with antihuman CD41-allophycocyanin, and CD41 þ MKs were separated using a FACS Aria cell sorter (BD Biosciences).After washing and centrifugation, 1 mL of Trizol (Life Technologies, Carlsbad, California, United States) was added and mixed thoroughly.After extracting total RNA, we used Nanodrop (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and Qiaxcel (QIAGEN, Hilden, Germany) to detect the concentration and purity of the extracted RNA.The samples of total RNA (1 µg) were treated with Ribo-off ribosomal RNA (rRNA) Depletion Kit (Vazyme, Nanjing, China) before the RNA-sequencing libraries were constructed.The RNA-sequencing libraries were prepared using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina following the manufacturer's instructions (Vazyme, Nanjing, China).Annotations of mRNA in the human genome were retrieved from the GENCODE V29 (https://www.Gencodegenes.org/human/re-lease_19.html).The genes that were differentially expressed between groups were analyzed using a t-test.The most differentially expressed genes were investigated for their involvement in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (https://www.genome.jp/kegg/)using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (https://david.ncifcrf.gov/).The enriched pathways were filtered with p-values <0.01.Preranked gene set enrichment analysis (GSEA) was run on the ranked list using the Molecular Signatures Database (MSigDB) (https://www.gseamsigdb.org/gsea/msigdb/)as the gene sets.

Quantitative Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from CD41 þ MKs using RNAiso Plus reagent (Takara, Shiga, Japan), and 1.5 µg total RNA from cultured cells was reverse transcribed using a PrimeScriptP RT Reagent Kit (Takara) according to the manufacturer's instructions.RT-qPCR was performed using a 7,500 Fast Real-Time PCR System (Applied Biosystems, Foster City, California, United States).The amplified transcript level of each specific gene was normalized to that of GAPDH.

Statistical Analysis
Statistical analysis was performed using SPSS statistics 24.0 (SPSS) and R statistical software.Results were expressed as means AE standard error.The Kolmogorov-Smirnov test was used to check for the normal distribution of data, and statistical differences between groups were observed using a t-test or the Mann-Whitney test.The Kaplan-Meier curve was performed and the log-rank test was applied to estimate and compare OS between the two groups.The level of significance was p < 0.05 for all analyses.

RNA-Sequencing of Megakaryocytes
We further explored the transcriptional heterogeneity of specific MKs status.Among 10,643 mRNA that were detected, 4,826 were differentially expressed in patients with MK mutation.Within these 4,826 mRNA, 269 were continuously upregulated and 4,557 continuously downregulated in group A. Gene ontology (GO) analyses suggested that the differentially expressed genes were associated with chromatin modification, DNA repair, transcriptional regulation, programmed cell death, and other important functions.Further investigation of these processes showed that platelet formation and function were the core processes of the GO tree (►Fig.3A).
Increased expression of MKs genes, not just in the intracellular proteins (PKM, VWF, and FLNA), but also cell surface antigens (CD36, ITGB1, and ITGB3), was noted.KEGG pathway analyses suggested that innate immune responses, RNA splicing, and mRNA processing were most enriched among the differentially expressed genes (►Fig.3B).GSEA analysis identified that platelet function, platelet aggregation, and platelet activation pathways were upregulated the most (►Fig.4).We focused on several candidate mRNA and verified the changes in their expression levels, and qRT-PCR assays showed that levels of CXCL10, CD62P, and S100A9 were remarkably increased (►Fig. 5).

Discussion
MDS is characterized by multiple clonal hematopoietic defects, and some patients may present with isolated thrombocytopenia and megakaryocytic dysmorphia or atypia.The development and differentiation from MKs to platelets are revealed to be a complex process that can be driven by a number of genes.With the advent of next-generation sequencing, an increasing number of genes associated with megakaryocytopoiesis have been elucidated.In our present study, the most frequent mutation in MDS included TUBB1, NBEAL2, and VWF gene.TUBB1 mutation was commonly found to disrupt the normal assembly of microtubules and contributed to the accumulation of DNA damage and genetic instability. 16,17NBEAL2 mutation was associated with a genetic disturbance of MK differentiation, with 36 to 65% of MKs containing neutrophils. 18,19VWF mutation resulted in a reduced number of platelets by MKs, the ectopic release of platelets in the BM, and the increased clearance of platelet-VWF complexes. 20Regardless of age and IPSS-R score, patients with MK mutation were at increased risk of developing thrombocytopenia and/or platelet dysfunction during their lifetime. 21,22Therefore, early recognition of MK mutation in MDS patients could permit appropriate treatment and adequate monitoring for disease progression.MK mutation was associated with high levels of CD34 on MK, which was likely a result of dysplastic maturation committed to the megakaryocytic lineage.Indeed, previous studies had documented that GATA1, TP53, and RUNX1 genes were related to the high-level expression of CD34 on MKs.][25] The enhanced emperipolesis in patients with a high percent of CD34 þ CD61 þ MKs was of interest.Petzold et al confirmed that neutrophils can "pluck" on MKs to tune platelet release in BM.In Pierre Cunin's model system, they demonstrated that neutrophil membranes transfer to MKs' demarcation membrane system during emperipolesis.7][28][29] Our data found that CD34 þ CD61 þ MKs with emperipolesis exhibited hyperreactivity and immature immunity, epitomized by increased CD62P, CXCL10, and S100A9 transcripts.Together these results implied a mechanistic role of emperipolesis on MK differentiation, and defective platelet formation maybe associated with "pathological emperipolesis" in MDS patients with high levels of CD34 þ CD61 þ MKs.
Bleeding complications, as a major cause of morbidity and mortality, are commonly seen in MDS patients.There may be bleeding episodes of varying severity, and there are variable platelet aggregation defects correlated with poor prognosis.A study of 75 MDS cases showed defective platelet activation and increased apoptotic platelets consistent with defective platelet production. 10Another study observed that ADP was one of the most common agonists with the platelet aggregation defect and confirmed that defective platelet aggregation was strongly related to MDS of worse prognosis. 11These results were consistent with our findings in patients with MK mutation, especially with high levels of CD34 þ CD61 þ MKs.These patients showed a decrease in ADP-induced platelet aggregation and an increase in platelet proactivation and apoptotic platelets.Managing bleeding due to dysfunctional platelets was based on general principles, and the most studied intervention was the transfusion of "normal" platelets. 30However, it may not be effective in major bleeding, for example, intracranial hemorrhage.To make matters worse, one study suggested that platelet transfusion was instead associated with higher rates of adverse events and death. 31In the present study, PTR was increased for MDS patients with MK mutation.Future studies will expand the sample size and focus more on exploring the relationship between MK mutation and the molecular etiology of PTR.
In summary, our study was performed to characterize a poor prognostic factor in MDS patients.We presented evidence that (1) the most common point mutation was TUBB1 p.(R307H) and p.(Q43P), followed by NBEAL2 p.(S2054F) and VWF p.(G1172V); (2) MK mutation was associated with the high percent of CD34 þ CD61 þ MKs; (3) platelet formation and platelet function were commonly affected by MK mutation; (4) CD62P, CXCL10, and S100A9 may be the potential targets in patients with MK mutation.Further studies are no doubt required to evaluate the molecular link between gene defects and platelet production and to establish any prognostic value.

Fig. 2 Fig. 3
Fig. 2 Platelet aggregation and activation.(A) Platelet aggregation function in myelodysplastic syndromes patients with or without megakaryocyte mutation.(B) The expression of CD62P and Annexin V on unstimulated platelet surface.(C) The expression of CD62P and Annexin V on stimulated platelet surface.